Chimeric antigen receptor dendritic cell (car-dc) for treatment of cancer

ABSTRACT

The current invention provides monocytic cells transfected with chimeric antigen receptor (CAR) to selectively home to tumors and upon homing differentiate into dendritic cells capable of activating immunity which is inhibitory to said tumor in one embodiment of the invention, monocytic cells are transfected with a construct encoding an antigen binding domain, a transcellular or structural domain, and an intracellular signaling domain. In one specific aspect of the invention, the antigen binding domain interacts with sufficient affinity to a tumor antigen, capable of triggering said intracellular domain to induce an activation signal to induce monocyte differentiation into DC.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/118,027 filed on Feb. 19, 2015, the contents of which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to the fields of genetics,immunology and medicine. The invention pertains to the field ofimmunotherapy, more specifically the invention pertains to theutilization of monocytes that have been manipulated to home to tumorcells and upon binding to tumor antigens differentiating into monocyteswith cytotoxic properties to tumors, or dendritic cells.

BACKGROUND OF THE INVENTION

The immune system possesses the power to cure cancers based on publishedreports of immunologically mediated spontaneous regressions, which havebeen document in colon, lung, melanoma, liver, breast. Intriguingly,spontaneous regression clinically, as well as in an animal model ofspontaneous regression, seems to be associated primarily withstimulation of the innate immune system, comprising of macrophages, NKcells, NKT cells and neutrophils. Despite the original promising ofimmunotherapy, which will be mentioned, the field has focused on theadaptive immune response, specifically stimulation of T and B cells, andonly recently has interest re-ignited in the innate immune system.

The use of the immune system to treat cancer is theoretically appealingdue to the possibility of low toxicity, immunological memory, andability to attack metastatic disease. Early studies suggested thatvaccination to tumor antigens and tumors themselves may be possible.Specifically, Prehn back in 1957, obtained murine tumors and exposedthem to irradiation to increase immunogenicity. When these tumors wereimplanted into animals they were rejected. Subsequent administration ofthe original tumors resulted in rejection of the tumors, thus suggestingthat tumor specific antigens exist, which can stimulate immunity,especially subsequent to addition of a cellular stress such asirradiation. Twenty years later, using the same system it wasdemonstrated that cytotoxic T cells infiltrated the tumors that wereimplanted after rejection of the radiation induced tumors, thusdemonstrating conclusively that rejection was immunologically mediated,despite the fact that the tumors were syngeneic. In humans, one of theoriginal observations of immunological response to neoplasia was inpatients with paraneoplastic disease in which immune response to breastcancer antigens results in a multiple sclerosis-like disease caused bycross reactive immunity to neural antigens that are found on the breastcancer. Specific identification of tumor antigens on a molecular basisled to the discovery that some of the antigens are either self-proteinsaberrantly expressed, or mutations of self proteins.

Originally observations were made in patients bearing metastaticmelanomas, and then subsequently in other tumors, that the tumors areinfiltrated with various immunological components. These tumorinfiltrating lymphocytes (TILS), contain populations of cells andindividual clones that demonstrate tumor specificity; they lyseautologous tumor cells but not natural killer targets, allogeneic tumorcells, or autologous fibroblasts.

By isolating and expanding TILs in vitro, and then molecularlyidentifying what they are responding to, a variety of the well-knowntumor agents have been discovered such as MAGE-1, and MAGE-3, GAGE-1,MART-1, Melan-A, gp100, gp75 (TRP-2), tyrosinase, NY-ESO-1, mutated p16,and beta catenin. It is interesting that in the case of some antigens,such as gp75, the peptide that elicits tumor rejection results fromtranslation of an alternative open reading frame of the same gene. Thus,the gp75 gene encodes two completely different polypeptides, gp75 as anantigen recognized by immunoglobulin G antibodies in sera from a patientwith cancer, and a 24-amino acid product as a tumor rejection antigenrecognized by T cells. Peptides used for immunization generally are 8-9amino acids which have been demonstrated to be displayed in associationwith class I MHC molecules for recognition by T cells, and tumor cellshave been shown to express these naturally processed epitopes.

Despite the intellectual appeal of peptide based cancer vaccines, theresponse rate has been disappointingly low. According to a review bySteven Rosenberg's group at the NIH, the rate of objective response outof 440 patients treated at his institute was a dismal 2.6%.

The ability to make a universal yet versatile system to generate T cellsthat are capable of recognizing various types of cancers has importantclinical implications for the use of T cell-based therapies, thisconcept was approach initially by Rosenberg's group in the ex vivoexpansion of tumor infiltrating lymphocytes. One current strategyincorporates the use of genetic engineering to express a chimericantigen receptor (CAR) on T cells. The extracellular domain of a typicalCAR consists of the V_(H) and V_(L) domains—single-chain fragmentvariable (scFv)—from the antigen binding sites of a monoclonal antibody.The scFv is linked to a flexible transmembrane domain followed by atyrosine-based activation motif such as that from CD3ζ. The so-calledsecond and third generation CARs include additional activation domainsfrom co-stimulatory molecules such as CD28 and CD137 (41BB) which serveto enhance T cell survival and proliferation. CAR T cells offer theopportunity to seek out and destroy cancer cells by recognizingtumor-associated antigens (TAA) expressed on their surface. As such, therecognition of a tumor cells occurs via an MHC-independent mechanism.

Various preclinical and early-phase clinical trials highlight theefficacy of CAR T cells to treat cancer patients with solid tumors andhematopoietic malignancies. Despite of the promise that CAR T cellsmight have in treating cancer patients there are several limitations tothe generalized clinical application of CAR T cells. First, since nosingle tumor antigen is universally expressed by all cancer types, scFvin CAR needs to be constructed for each tumor antigen to be targeted.Second, the financial cost and labor-intensive tasks associated withidentifying and engineering scFvs against a variety of tumor antigensposes a major challenge. Third, tumor antigens targeted by CAR could bedown-regulated or mutated in response to treatment resulting in tumorevasion. Since current CAR T cells recognize only one target antigen,such changes in tumors negate the therapeutic effects. Therefore, thegeneration of CAR T cells capable of recognizing multiple tumor antigensis highly desired. Finally, CAR T cells react with target antigen weaklyexpressed on non-tumor cells, potentially causing severe adverseeffects. To avoid such “on-target off-tumor” reaction, use of scFvs withhigher specificity to tumor antigen is required. And although ongoingstudies are focused on generating methods to shut off CAR T cells invivo this system has yet to be developed and might pose additionalinherent challenges.

The current patent seeks to apply chimeric antigen receptor technologyto activation of monocytes, which naturally home into tumors, todifferentiated intratumorally said monocytes into dendritic cells whichare capable of antigen presentation, as well as direct killing oftumors.

DETAILED DESCRIPTION OF THE INVENTION

Chimeric antigen receptor (CAR) cellular therapeutics haverevolutionized the treatment of B cell malignancies achieving stunningsuccess rates. Unfortunately, solid tumors have yet to benefit from thistreatment. Additionally, patients treated with CAR-T cells lack B cellsfor the rest of their lives, as well as having the possibility of tumorlysis syndrome. This is in part due to the permanence of the CAR-T cellsin the patients after treatment. The current invention applies the useof CAR technology to monocytes with the purpose of inducingdifferentiation to dendritic cells (DC) subsequent to contact with tumorantigens. Given that monocytes have a fixed mitotic index, fears ofpermanent manipulation of the host are diminished.

“Treating a cancer”, “inhibiting cancer”, “reducing cancer growth”refers to inhibiting or preventing oncogenic activity of cancer cells.Oncogenic activity can comprise inhibiting migration, invasion, drugresistance, cell survival, anchorage-independent growth,non-responsiveness to cell death signals, angiogenesis, or combinationsthereof of the cancer cells.

The terms “cancer”, “cancer cell”, “tumor”, and “tumor cell” are usedinterchangeably herein and refer generally to a group of diseasescharacterized by uncontrolled, abnormal growth of cells (e.g., aneoplasia). In some forms of cancer, the cancer cells can spread locallyor through the bloodstream and lymphatic system to other parts of thebody (“metastatic cancer”).

“Ex vivo activated lymphocytes”, “lymphocytes with enhanced antitumoractivity” and “dendritic cell cytokine induced killers” are terms usedinterchangeably to refer to composition of cells that have beenactivated ex vivo and subsequently reintroduced within the context ofthe current invention. Although the word “lymphocyte” is used, this alsoincludes heterogenous cells that have been expanded during the ex vivoculturing process including dendritic cells, NKT cells, gamma delta Tcells, and various other innate and adaptive immune cells.

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in animals, including leukemias, carcinomas andsarcomas. Examples of cancers are cancer of the brain, melanoma,bladder, breast, cervix, colon, head and neck, kidney, lung, non-smallcell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus andMedulloblastoma.

The term “leukemia” is meant broadly progressive, malignant diseases ofthe hematopoietic organs/systems and is generally characterized by adistorted proliferation and development of leukocytes and theirprecursors in the blood and bone marrow. Leukemia diseases include, forexample, acute nonlymphocytic leukemia, chronic lymphocytic leukemia,acute granulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia,Schilling's leukemia, stem cell leukemia, subleukemic leukemia,undifferentiated cell leukemia, hairy-cell leukemia, hemoblasticleukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cellleukemia, acute monocytic leukemia, leukopenic leukemia, lymphaticleukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenousleukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cellleukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocyticleukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, and promyelocytic leukemi.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues, and/orresist physiological and non-physiological cell death signals and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrmcous carcinoma, carcinomavillosum, carcinoma gigantocellulare, glandular carcinoma, granulosacell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma, molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, and carcinoma scroti.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar, heterogeneous, orhomogeneous substance. Sarcomas include, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma,liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoidsarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilns'tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, andtelangiectaltic sarcoma. Additional exemplary neoplasias include, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, breast cancer; ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid,premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, cervical cancer, endometrial cancer, and adrenal corticalcancer.

In some particular embodiments of the invention, the cancer treated is amelanoma. The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas include, forexample, Harding-Passey melanoma, juvenile melanoma, lentigo malignamelanoma, malignant melanoma, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,nodular melanoma subungal melanoma, and superficial spreading melanoma.

The term “polypeptide” is used interchangeably with “peptide”, “alteredpeptide ligand”, and “flourocarbonated peptides.”

The term “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

The term “T cell” is also referred to as T lymphocyte, and means a cellderived from thymus among lymphocytes involved in an immune response.The T cell includes any of a CD8-positive T cell (cytotoxic T cell;CTL), a CD4-positive T cell (helper T cell), a suppressor T cell, aregulatory T cell such as a controlling T cell, an effector cell, anaive T cell, a memory T cell, an αβT cell expressing TCR α and βchains, and a γδ T cell expressing TCR γ and δ chains. The T cellincludes a precursor cell of a T cell in which differentiation into a Tcell is directed.

Examples of “cell populations containing T cells” include, in additionto body fluids such as blood (peripheral blood, umbilical blood etc.)and bone marrow fluids, cell populations containing peripheral bloodmononuclear cells (PBMC), hematopoietic cells, hematopoietic stem cells,umbilical blood mononuclear cells etc., which have been collected,isolated, purified or induced from the body fluids. Further, a varietyof cell populations containing T cells and derived from hematopoieticcells can be used in the present invention. These cells may have beenactivated by cytokine such as IL-2 in vivo or ex vivo. As these cells,any of cells collected from a living body, or cells obtained via ex vivoculture, for example, a T cell population obtained by the method of thepresent invention as it is, or obtained by freeze preservation, can beused.

The term “antibody” is meant to include both intact molecules as well asfragments thereof that include the antigen-binding site. Whole antibodystructure is often given as H₂L₂ and refers to the fact that antibodiescommonly comprise 2 light (L) amino acid chains and 2 heavy (H) aminoacid chains. Both chains have region's capable of interacting with astructurally complementary antigenic target. The regions interactingwith the target are referred to as “variable” or “V” regions and arecharacterized by differences in amino acid sequence from antibodies ofdifferent antigenic specificity. The variable regions of either H or Lchains contains the amino acid sequences capable of specifically bindingto antigenic targets. Within these sequences are smaller sequencesdubbed “hypervariable” because of their extreme variability betweenantibodies of differing specificity. Such hypervariable regions are alsoreferred to as “complementarity determining regions” or “CDR” regions.These CDR regions, account for the basic specificity of the antibody fora particular antigenic determinant structure. The CDRs representnon-contiguous stretches of amino acids within the variable regions but,regardless of species, the positional locations of these critical aminoacid sequences within the variable heavy and light chain regions havebeen found to have similar locations within the amino acid sequences ofthe variable chains. The variable heavy and light chains of allantibodies each have 3 CDR regions, each non-contiguous with the others(termed L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy(H) chains. The antibodies disclosed according to the invention may alsobe wholly synthetic, wherein the polypeptide chains of the antibodiesare synthesized and, possibly, optimized for binding to the polypeptidesdisclosed herein as being receptors. Such antibodies may be chimeric orhumanized antibodies and may be fully tetrameric in structure, or may bedimeric and comprise only a single heavy and a single light chain.

The term “effective amount” or “therapeutically effective amount” meansa dosage sufficient to treat, inhibit, or alleviate one or more symptomsof a disease state being treated or to otherwise provide a desiredpharmacologic and/or physiologic effect, especially enhancing T cellresponse to a selected antigen. The precise dosage will vary accordingto a variety of factors such as subject-dependent variables (e.g., age,immune system health, etc.), the disease, and the treatment beingadministered.

The terms “individual”, “host”, “subject”, and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, primates, for example, human beings, as well as rodents,such as mice and rats, and other laboratory animals.

As used herein, the term “treatment regimen” refers to a treatment of adisease or a method for achieving a desired physiological change, suchas increased or decreased response of the immune system to an antigen orimmunogen, such as an increase or decrease in the number or activity ofone or more cells, or cell types, that are involved in such response,wherein said treatment or method comprises administering to an animal,such as a mammal, especially a human being, a sufficient amount of twoor more chemical agents or components of said regimen to effectivelytreat a disease or to produce said physiological change, wherein saidchemical agents or components are administered together, such as part ofthe same composition, or administered separately and independently atthe same time or at different times (i.e., administration of each agentor component is separated by a finite period of time from one or more ofthe agents or components) and where administration of said one or moreagents or components achieves a result greater than that of any of saidagents or components when administered alone or in isolation.

The term “anergy” and “unresponsiveness” includes unresponsiveness to animmune cell to stimulation, for example, stimulation by an activationreceptor or cytokine. The anergy may occur due to, for example, exposureto an immune suppressor or exposure to an antigen in a high dose. Suchanergy is generally antigen-specific, and continues even aftercompletion of exposure to a tolerized antigen. For example, the anergyin a T cell and/or NK cell is characterized by failure of production ofcytokine, for example, interleukin (IL)-2. The T cell anergy and/or NKcell anergy occurs in part when a first signal (signal via TCR or CD-3)is received in the absence of a second signal (costimulatory signal)upon exposure of a T cell and/or NK cell to an antigen.

The term “enhanced function of a T cell”, “enhanced cytotoxicity” and“augmented activity” means that the effector function of the T celland/or NK cell is improved. The enhanced function of the T cell and/orNK cell, which does not limit the present invention, includes animprovement in the proliferation rate of the T cell and/or NK cell, anincrease in the production amount of cytokine, or an improvement incytotoxity. Further, the enhanced function of the T cell and/or NK cellincludes cancellation and suppression of tolerance of the T cell and/orNK cell in the suppressed state such as the anergy (unresponsive) state,or the rest state, that is, transfer of the T cell and/or NK cell fromthe suppressed state into the state where the T cell and/or NK cellresponds to stimulation from the outside.

The term “expression” means generation of mRNA by transcription fromnucleic acids such as genes, polynucleotides, and oligonucleotides, orgeneration of a protein or a polypeptide by transcription from mRNA.Expression may be detected by means including RT-PCR, Northern Blot, orin situ hybridization.

“Suppression of expression” refers to a decrease of a transcriptionproduct or a translation product in a significant amount as comparedwith the case of no suppression. The suppression of expression hereinshows, for example, a decrease of a transcription product or atranslation product in an amount of 30% or more, preferably 50% or more,more preferably 70% or more, and further preferably 90% or more.

In one embodiment of the invention the CAR-DC are antigen-loaded andco-cultured with T-lymphocytes to produce antigen-specific T-cells. Asused herein, the term “antigen-specific T-cells” refers to T-cells thatproliferate upon exposure to the antigen-loaded APCs of the presentinvention, as well as to develop the ability to attack cells having thespecific antigen on their surfaces. Such T-cells, e.g., cytotoxicT-cells, lyse target cells by a number of methods, e.g., releasing toxicenzymes such as granzymes and perforin onto the surface of the targetcells or by effecting the entrance of these lytic enzymes into thetarget cell interior, Generally, cytotoxic T-cells express CD8 on theircell surface. T-cells that express the CD4 antigen CD4, commonly knownas “helper” T-cells, can also help promote specific cytotoxic activityand may also be activated by the antigen-loaded APCs of the presentinvention. In certain embodiments, the cancer cells, the APCs and eventhe T-cells can be derived from the same donor whose MNC yielded the DC,which can be the patient or an HLA—or obtained from the individualpatient that is going to be treated. Alternatively, the cancer cells,the APCs and/or the T-cells can be allogeneic.

The invention provides means of inducing an anti-cancer response in amammal, comprising the steps of initially “priming” the mammal byadministering an agent that causes local accumulation of CAR-DC.Subsequently, a tumor antigen is administered in the local area wheresaid agents causing accumulation of antigen presenting cells isadministered. A time period is allowed to pass to allow for said antigenpresenting cells to traffic to the lymph nodes. Subsequently amaturation signal, or a plurality of maturation signals are administeredto enhance the ability of said antigen presenting cell to activateadaptive immunity. In some embodiments of the invention activators ofadaptive immunity are concurrently given, as well as inhibitors of thetumor derived inhibitors are administered to derepress the immunesystem.

In one embodiment priming of the patient is achieved by administrationof GM-CSF subcutaneously in the area in which antigen is to be injected.Various scenarios are known in the art for administration of GM-CSFprior to administration, or concurrently with administration of antigen.The practitioner of the invention is referred to the followingpublications for dosage regimens of GM-CSF and also of peptide antigens.

Subsequent to priming, the invention calls for administration of tumorantigen. Various tumor antigens may be utilized, in one preferredembodiment, lysed tumor cells from the same patient area utilized, Meansfor generation of lysed tumor cells are well known in the art anddescribed in the following references. One example method for generationof tumor lysate involves obtaining frozen autologous samples which areplaced in hanks buffered saline solution (HBSS) and gentamycin 50 μg/mlfollowed by homogenization by a glass homogenizer. After repeatedfreezing and thawing, particle-containing samples are selected andfrozen in aliquots after radiation with 25 kGy. Quality assessment forsterility and endotoxin content is performed before freezing. Celllysates are subsequently administered into the patient in a preferredmanner subcutaneously at the local areas where DC priming was initiated.After 12-72 hours, the patient is subsequently administered with anagent capable of inducing maturation of DC. Agents useful for thepractice of the invention, in a preferred embodiment include BCG andHMGB1 peptide. Other useful agents include: a) histone DNA; b) imiqimod;c) beta-glucan; d) hsp65; e) hsp90; f) HMGB-1; g) lipopolysaccharide; h)Pam3CSK4; i) Poly I: Poly C; j) Flagellin; k) MALP-2; l)Imidazoquinoline; m) Resiquimod; n) CpG oligonucleotides; o) zymosan; p)peptidoglycan; q) lipoteichoic acid; r) lipoprotein from gram-positivebacteria; s) lipoarabinomannan from mycobacteria; t)Polyadenylic-polyuridylic acid; u) monophosphoryl lipid A; v) singlestranded RNA; w) double stranded RNA; x) 852A; y) rintatolimod; z)Gardiquimod; and aa) lipopolysaccharide peptides. The procedure isperformed in a preferred embodiment with the administration of IDOsilencing siRNA or shRNA containing the effector sequences a)UUAUAAUGACUGGAUGUUC; b) GUCUGGUGUAUGAAGGGUU; c) CUCCUAUUUUGGUUUAUGC andd) GCAGCGUCUUUCAGUGCUU. siRNA or shRNA may be administered throughvarious modalities including biodegradable matrices, pressure gradientsor viral transfect. In another embodiment, autologous dendritic cellsare generated and IDO is silenced, prior to, concurrent with orsubsequent to silencing, said dendritic cells are pulsed with tumorantigen and administered systemically.

In one embodiment of the invention mature DC are modified with CARtransfection prior to administration. Culture of dendritic cells is wellknown in the art, for example, U.S. Pat. No. 6,936,468, issued toRobbins, et al., for the use of tolerogenic dendritic cells forenhancing tolerogenicity in a host and methods for making the same.Although the current invention aims to reduce tolerogenesis, theessential means of dendritic cell generation are disclosed in thepatent. U.S. Pat. No. 6,734,014, issued to Hwu, et al., for methods andcompositions for transforming dendritic cells and activating T cells.Briefly, recombinant dendritic cells are made by transforming a stemcell and differentiating the stem cell into a dendritic cell. Theresulting dendritic cell is said to be an antigen presenting cell whichactivates T cells against MHC class I-antigen targets. Antigens for usein dendritic cell loading are taught in, e.g., U.S. Pat. No. 6,602,709,issued to Albert, et al. This patent teaches methods for use ofapoptotic cells to deliver antigen to dendritic cells for induction ortolerization of T cells. The methods and compositions are said to beuseful for delivering antigens to dendritic cells that are useful forinducing antigen-specific cytotoxic T lymphocytes and T helper cells.The disclosure includes assays for evaluating the activity of cytotoxicT lymphocytes. The antigens targeted to dendritic cells are apoptoticcells that may also be modified to express non-native antigens forpresentation to the dendritic cells. The dendritic cells are said to beprimed by the apoptotic cells (and fragments thereof) capable ofprocessing and presenting the processed antigen and inducing cytotoxic Tlymphocyte activity or may also be used in vaccine therapies. U.S. Pat.No. 6,455,299, issued to Steinman, et al., teaches methods of use forviral vectors to deliver antigen to dendritic cells. Methods andcompositions are said to be useful for delivering antigens to dendriticcells, which are then useful for inducing T antigen specific cytotoxic Tlymphocytes. The disclosure provides assays for evaluating the activityof cytotoxic T lymphocytes. Antigens are provided to dendritic cellsusing a viral vector such as influenza virus that may be modified toexpress non-native antigens for presentation to the dendritic cells. Thedendritic cells are infected with the vector and are said to be capableof presenting the antigen and inducing cytotoxic T lymphocyte activityor may also be used as vaccines.

Immune cells for use in the practice of the invention include DCs, thepresence of which may be checked in the previously described method, arepreferably selected from myeloid cells (such as monocytic cells andmacrophages) expressing langerin, MHC (major histocompatibility complex)class II, CCR2 (chemokine (C—C motif) receptor 2), CX3CR1 and/or Gr1molecules in mice; myeloid cells expressing CD14, CD16, HLA dR (humanleukocyte antigen disease resistance) molecule, langerin, CCR2 and/orCX3CR1 in humans; dendritic cells expressing CD11c, MHC class itmolecules, and/or CCR7 molecules; and IL-1β producing dendritic cells.CD8 T cells, the presence of which may be checked in the previouslydescribed method, are preferably selected from CD3+, CD4+ and/or CD8+ Tlymphocytes, FOXP3 (forkhead box P3) T lymphocytes, Granzyme B/TIA(Tcell-restricted intracellular antigen) T lymphocytes, and Tel cells(IFN-.gamma. producing CD8+T lymphocytes). Immune cells expressing aprotein that binds calreticulin, such immune cells may be selected fromcells expressing at least one of the following proteins; LRP1 (Lowdensity lipoprotein receptor-related protein 1, CD91), Ca.sup.++-bindingproteins such as SCARF1 and SCARF2, MSR1 (Macrophage scavenger receptor1), SRA, CD59 (protectin), CD207 (langerin), and THSD1 (thrombospondin).There are numerous means known in the art to identify cells expressingvarious antigens, these include immunochemistry, immunophenotyping, flowcytometry, Elispots assays, classical tetramer staining, andintracellular cytokine stainings.

Macrophages selectively phagocytose tumor cells, but this process iscountered by protective molecules on tumor cells such as CD47, whichbinds macrophage signal-regulatory protein α to inhibit phagocytosis.Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Inone embodiment of the invention CAR-MSC are administered together withan agent that blocks CD47 activity. It has been demonstrated thatactivation of TLR signaling pathways in macrophages synergizes withblocking CD47 on tumor cells to enhance tumor phagocytosis. Bruton'styrosine kinase (Btk) mediates TLR signaling in macrophages.Calreticulin, previously shown to be a protein found on cancer cellsthat activated macrophage phagocytosis of tumors, is activated inmacrophages for secretion and cell-surface exposure by TLR and Btk totarget cancer cells for phagocytosis, even if the cancer cellsthemselves do not express calreticulin. In one embodiment of theinvention TLR agonists are administered that stimulate expression ofcalreticulin and/or enhance macrophage phagocytosis of tumors.

IL-27 induces macrophage ability to kill tumor cells in vitro and invivo, as well as altering the tumor promoting M2/myeloid suppressorcells into tumoricidal cells. In one embodiment of the inventionaddition of IL-27 or compounds capable of activating the IL-27 receptorsignaling are administered together with IL-27 to enhance tumorphagocytosis by macrophages.

Tumor-associated macrophages, deriving from monocytes or migrating intothe tumor, are an important constituent of tumor microenvironments,which in many cases modulates tumor growth, tumor angiogenesis, immunesuppression, metastasis and chemoresistance. Mechanisms of macrophagepromotion of tumor growth include production of EGF, M-CSF, VEGF.

Macrophage infiltration of tumors is associated with poor prognosis inrenal, melanoma, breast, pancreatic, lung, endometrial, bladder,prostate.

Tumor growth are inhibited when monocytes/macrophages are ablated. Thereis ample evidence that many anticancer modalities currently used in theclinic have unique and distinct properties that modulate therecruitment, polarization and tumorigenic activities of macrophages inthe tumor microenvironments, By manipulating tumor-associatedmacrophages significant impact on the clinical efficacies of andresistance to these anticancer modalities. Accordingly, in one aspect ofthe invention, CAR-DC, CAR-monocytes, or CAR-macrophages are utilized toforce the tumor microenvironment to stimulate tumor killing and inhibitmacrophage or macrophage related cells from promoting tumor growth.Within the context of the invention, the use of drugs targetingtumor-associated macrophages, especially c-Fms kinase inhibitors andhumanized antibodies targeting colony-stimulating factor-1 receptor, areenvisioned.

Tumors mediate various effects to reprogram macrophages, these areusually mediated via IL-10 and other cytokines such as VEGF, TGF-beta,and M CSF, which cause macrophages to lose tumor cytotoxicity and shiftinto tumor promoting, immune suppressive, angiogenic supporting cells.Related to tumor manipulated monocytes are myeloid derived suppressorcells, which are similar to myeloid progenitor cells, or the previouslydescribed “natural suppressor” cell.

Irradiated tissues induce a TLR-1 reprogramming of macrophages topromote tumor growth and angiogenesis. Macrophage promotion of tumorgrowth is seen in numerous situations, in one example, treating of tumorbearing animals with BRAF inhibitors results in upregulation ofmacrophage production of VEGF which accelerates tumor growth.Mechanistically, it is known that tumors produce factors such as GM-CSFwhich in pant stimulate macrophages to produce CCL18, which promotestumor metastasis. Additionally, the lactic acid microenvironment of thetumor has been shown to promote skewing of macrophages towards attumor-promoting M2 type. It has been shown that lactic acid produced bytumour cells, as a by-product of aerobic or anaerobic glycolysis,possesses an essential role in inducing the expression of VEGF and theM2-like polarization of tumour-associated macrophages, specificallyinducing expression of arginase 1 through a HIF-1alpha dependentpathway. Mechanistically, it is known that lactic acid in tumors isgenerated in a large part by lactate dehydrogenase-A (LDH-A), whichconverts pyruvate to lactate. siRNA silencing of LDH-A in Pan02pancreatic cancer cells that are injected in C57BL/6 mice results indevelopment of smaller tumors than mice injected with wild type,non-silenced Pan02 cells. Associated with the reduced tumor growth wereobservations of a decrease in the frequency of myeloid-derivedsuppressor cells (MDSCs) in the spleens of mice carrying LDH-A-silencedtumors. NK cells from LDH-A-depleted tumors had improved cytolyticfunction. Exogenous lactate administration was shown to increase thefrequency of MDSCs generated from mouse bone marrow cells with GM-CSFand IL-6 in vitro. Furthermore lactate pretreatment of NK cells in vitroinhibited cytolytic function of both human and mouse NK cells. Thisreduction of NK cytotoxic activity was accompanied by lower expressionof perforin and granzyme in NK cells. The expression of NKp46 was lowerin lactate-treated NK cells. Accordingly, in one embodiment of theinvention, depletion of glucose levels using a ketogenic diet to lowerlactate production by glycolytic tumors is utilized to augmenttherapeutic effects of CAR-DC. Utilization of ketogenic diet has beenpreviously described for immune modulation, and cancer therapy. Specificquantification of intratumoral lactate and its manipulation has beendescribed and incorporated by reference. Potentiation ofchemotherapeutic and radiotherapeutic effects by ketogenic diets havebeen reported and techniques are incorporated by reference for use withthe current CAR-DC invention. Suppression of tumor growth and activityinduced by ketogenic diet may be augmented by addition of hyperbaricoxygen, thus in one embodiment of the invention, the utilization ofoxidative therapies, as disclosed in references incorporated, togetherwith ketogenic diet is utilized to augment therapeutic efficacy ofCAR-DC.

Not only has it been well known that monocytes and macrophagesinfiltrate tumors and appear to support tumor growth through growthfactor production and secretion of angiogenic agents, but suggestionshave been made that tumors themselves, as part of the epithelialmesenchymal transition may actually differentiate into monocytes in partassociated with TGF-beta production. Specifically, a study reportedperforming gene-profiling analysis of mouse mammary EpRas tumor cellsthat had been allowed to adopt an epithelial to mesenchymal transitionprogram after long-term treatment with TGF-β1 for 2 weeks. While thetreated cells acquired traits of mesenchymal cell differentiation andmigration, gene analysis revealed another cluster of induced genes,which was specifically enriched in monocyte-derived macrophages, mastcells, and myeloid dendritic cells, but less in other types of immunecells. Further studies revealed that this monocyte/macrophage genecluster was enriched in human breast cancer cell lines displaying an EMTor a Basal B profile, and in human breast tumors with EMT andundifferentiated (ER−/PR−) characteristics. The plasticity of tumorcells to potentially monocytic lineages should come as no surprise giventhat tumor cells have been shown to differentiate directly intopericytes, and endothelial cells/vascular channels.

Dopamine possesses antiangiogenic effects as well as myeloprotectiveeffects, in one embodiment of the invention addition of dopamine to theCAR-DC treatment is disclosed.

Vinblastine is a widely used chemotherapeutic agent that has beendemonstrated to induce dendritic cell maturation. In one embodiment ofthe invention CAR-DC are utilized together with vinblastine therapy toinduce augmented anticancer activity. Oxiplatin and anthracyclines havebeen demonstrated to not only directly kill tumor cells but alsostimulate T cell immunity against tumor cells. It was demonstrated thatthese agents induce a rapid and prominent invasion of interleukin(IL)-17-producing γδ (Vγ4(+) and Vγ6(+)) T lymphocytes (γδ T17 cells)that precedes the accumulation of CD8 CTLs within the tumor bed. In Tcell receptor δ(−/−) or Vγ4/6(−/−) mice, the therapeutic efficacy ofchemotherapy was reduced and furthermore no IL-17 was produced bytumor-infiltrating T cells, and CD8 CTLs did not invade the tumor aftertreatment. Although γδ Th17 cells could produce both IL-17A and IL-22,the absence of a functional IL-17A-IL-17R pathway significantly reducedtumor-specific T cell responses elicited by tumor cell death, and theefficacy of chemotherapy in four independent transplantable tumormodels. The adoptive transfer of γδ T cells to naïve mice restored theefficacy of chemotherapy in IL-17A(−/−) hosts. The anticancer effect ofinfused γδ T cells was lost when they lacked either IL-1R1 or IL-17A.

Intratumoral injection of dendritic cells stimulates antitumor immunityin vivo in clinical situations, suggesting that modulating the antigenpresenting cell in the tumor microenvironment will induce an antitumorresponse. Administration of radiotherapy to tumors to induce immunogeniccell death, followed by intratumoral administration of DC has beendemonstrated to result in enhanced antigen presentation, accordingly,this technique may be modified to enhance effects of CAR-DC. Theinduction of immunity to tumors in the present invention is associatedwith the unique nature of: a) ongoing basal cell death within the tumor;and b) cell death induced by chemotherapy, radiotherapy, hyperthermia,or otherwise induced cell death. Cell death can be classified accordingto the morphological appearance of the lethal process (that may beapoptotic, necrotic, autophagic or associated with mitosis),enzymological criteria (with and without the involvement of nucleases ordistinct classes of proteases, like caspases), functional aspects(programmed or accidental, physiological or pathological) orimmunological characteristics (immunogenic or non-immunogenic). Celldeath is defined as “immunogenic” or “immune stimulatory” if dying cellsthat express a specific antigen (for example a tumor associated antigen,phosphotidyl serine, or calreticulin), yet are uninfected (and hencelack pathogen-associated molecular patterns), and are injectedsubcutaneously into mice, in the absence of any adjuvant, cause aprotective immune response against said specific antigen. Such aprotective immune response precludes the growth of living transformedcells expressing the specific antigen injected into mice. When cancercells succumb to an immunogenic cell death (or immunogenic apoptosis)modality, they stimulate the immune system, which then mounts atherapeutic anti-cancer immune response and contributes to theeradication of residual tumor cells. Conversely, when cancer cellssuccumb to a non-immunogenic death modality, they fail to elicit such aprotective immune response. Regardless of the types of cell death thatare ongoing, the tumor derived immune suppressive molecules contributeto general inhibition or inability of the tumor to be eliminated.

Within the practice of the invention, CAR-DC are administeredconcurrently, prior to, or subsequent to administration of an agent thatinduces immunogenic cell death in a patient. Methods of determiningwhether compounds induce immunogenic cell death are known in the art andinclude the following, which was described by Zitvogel et al. (a)treating the cells, the mammalian cells and inducing the cell death orapoptosis, typically of mammalian cancer cells capable of expressingcalreticulin (CRT), by exposing said mammalian cells to a particulardrug (the test drug), for example 18 hours; (b) inoculating (for exampleintradermally) the dying mammalian cells from step (a) in a particulararea (for example a flank) of the mammal, typically a mouse, to inducean immune response in this area of the mammal; (c) inoculating (forexample intradermally) the minimal tumorigenic dose of syngeneic livetumor cells in a distinct area (for example the opposite flank) from thesame mammal, for example 7 days after step (b); and (d) comparing thesize of the tumor in the inoculated mammal with a control mammal alsoexposed to the minimal tumorigenic dose of syngeneic live tumor cells ofstep (c) [for example a mouse devoid of T lymphocyte], the stabilizationor regression of the tumor in the inoculated mammal being indicative ofthe drug immunogenicity. Other in vitro means are available forassessing the ability of various drugs or therapeutic approaches toinduce immunogenic cell death. Specific characteristics to assess whenscreening for immunogenic cell death include: a) ability to inducedendritic cell maturation in vitro; b) ability to activate NK cells; andc) ability to induce activation of gamma delta T cells or NKT cells.Specific drugs known to induce immunogenic cell death include oxiplatineand anthracyclines, as well as radiotherapy, and hyperthermia. In thecase of chemotherapies, certain chemotherapies that activate TLR4through induction of HMGB1 have been observed to function suboptimallyin patients that have a TLR4 polymorphism, thus suggesting actualcontribution of TLR activation as a means of chemotherapy inhibition ofcancer. Additionally, oncoviruses or oncolytic viruses are known toinduce immunogenic cell death and may be useful for the practice of theinvention.

The CAR-DC disclosed in the invention may be utilized in combinationwith conventional immune modulators including BCG, CpG DNA, interferonalpha, tumor bacterial therapy, checkpoint inhibitors, Treg depletingagents, and low dose cyclophosphamide.

In one embodiment of the invention CAR-DC cells are generated withspecificity towards ROBO-4. Numerous means of generating CAR-T cells areknown in the art, which are applied to CAR-DC. In one embodiment of theinvention FMC63-28z CAR (Genebank identifier HM852952.1), is used as thetemplate for the CAR except the anti-CD19, single-chain variablefragment sequence is replaced with an ROBE-4 fragment. The construct issynthesized and inserted into a pLNCX retroviral vector. Retrovirusesencoding the ROBO-4-specific CAR are generated using the retroviruspackaging kit, Ampho (Takara), following the manufacturer's protocol.For generation of CAR-DC cells donor blood is obtained and aftercentrifugation on Ficoll-Hypaque density gradients (Sigma-Aldrich),PBMCs are plated at 2×10(6) cells/mL in cell culture for 2 hours and theadherent cells are collected. The cells were then stimulated for 2 dayson a tissue-culture-treated 24-well plate containing M-CSF at aconcentration of 100 ng/ml For retrovirus transduction, a 24-well plateare coated with RetroNectin (Takara) at 4° C. overnight, according tothe manufacturer's protocol, and then blocked with 2% BSA at roomtemperature for 30 min. The plate was then loaded with retrovirussupernatants at 300 μL/well and incubated at 37° C. for 6 h. Next,1×10(6) stimulated adherent cells in 1 mL of medium are added to 1 mL ofretrovirus supernatants before being transferred to the pre-coated wellsand cultured at 37° C. for 2 d. The cells are then transferred to atissue-culture-treated plate at 1×10 (6) cells/mL and cultured in thepresence of 100 U/mL of recombinant human M-CSF, applying the T cellprotocol but not, utilizing IL-2 or antiCD3/antiCD28.

Other means of generating CARs are known in the art and incorporated byreference. For example, Groner's group genetically modified Tlymphocytes and endowed them with the ability to specifically recognizecancer cells. Tumor cells overexpressing the ErbB-2 receptor served as amodel. The target cell recognition specificity was conferred to Tlymphocytes by transduction of a chimeric gene encoding the zeta-chainof the TCR and a single chain antibody (scFv(FRP5)) directed against thehuman ErbB-2 receptor. The chimeric scFv(FRP5)-zeta gene was introducedinto primary mouse T lymphocytes via retroviral gene transfer. Naive Tlymphocytes were activated and infected by cocultivation with aretrovirus-producing packaging cell line. The scFv(FRP5)-zeta fusiongene was expressed in >75% of the T cells. These T cells lysedErbB-2-expressing target cells in vitro with high specificity. In asyngeneic mouse model, mice were treated with autologous, transduced Tcells. The adoptively transferred scFv(FRP5)-zeta-expressing T cellscaused total regression of ErbB-2-expressing tumors. The presence of thetransduced T lymphocytes in the tumor tissue was monitored. No humoralresponse directed against the transduced T cells was observed. Absdirected against the ErbB-2 receptor were detected upon tumor lysis.Hombach et al. constructed an anti-CEA chimeric receptor whoseextracellular moiety is composed of a humanized scFv derived from theanti-CEA mAb BW431/26 and the CH2/CH3 constant domains of human IgG. Theintracellular moiety consists of the gamma-signaling chain of the humanFc epsilon RI receptor constituting a completely humanized chimericreceptor. After transfection, the humBW431/26 scFv-CH2CH3-gamma receptoris expressed as a homodimer on the surface of MD45 T cells.Co-incubation with CEA+ tumor cells specifically activates grafted MD45T cells indicated by IL-2 secretion and cytolytic activity against CEA+tumor cells. Notably, the efficacy of receptor-mediated activation isnot affected by soluble CEA up to 25 micrograms/ml demonstrating theusefulness of this chimeric receptor for specific cellular activation bymembrane-bound CEA even in the presence of high concentrations of CEA,as found in patients during progression of the disease (200). Thesemethods are described to guide one of skill in the art to practicing theinvention, which in one embodiment is the utilization of CAR T cellapproaches towards targeting tumor endothelium as comparted to simplytargeting the tumor itself.

Targeting of mucins associated with cancers has been performed with CART cells by grafting the antibody that binds to the mucin with CD3 zetachain. For the purpose of the invention, this procedure is modified forCAR-DC. In an older publication chimeric immune receptor consisting ofan extracellular antigen-binding domain derived from the CC49 humanizedsingle-chain antibody, linked to the CD3zeta signaling domain of the Tcell receptor, was generated (CC49-zeta). This receptor binds to TAG-72,a mucin antigen expressed by most human adenocarcinomas. CC49-zeta wasexpressed in CD4+ and CD8+ T cells and induced cytokine production onstimulation. Human T cells expressing CC49-zeta recognized and killedtumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta Tcells did not mediate bystander killing of TAG-72-negative cells. Inaddition, CC49-zeta T cells not only killed FasL-positive tumor cells invitro and in vivo, but also survived in their presence, and wereimmunoprotective in intraperitoneal and subcutaneous murine tumorxenograft models with TAG-72-positive human tumor cells. Finally,receptor-positive T cells were still effective in killingTAG-72-positive targets in the presence of physiological levels ofsoluble TAG-72, and did not induce killing of TAG-72-negative cellsunder the same conditions.

For clinical practice of the invention several reports exist in the artthat would guide the skilled artisan as to concentrations, cell numbers,and dosing protocols useful. While in the art CAR T cells have beenutilized targeting surface tumor antigens, the main issue with thisapproach is the difficulty of T cells to enter tumors due to featuresspecific to the tumor microenvironment. These include higherinterstitial pressure inside the tumor compared to the surroundings,acidosis inside the tumor, and expression in the tumor of FasL whichkills activated T cells. Accordingly the invention seeks to moreeffectively utilize CAR-DC cells by directly targeting them to tumorendothelium, which is in direct contact with blood and therefore notsusceptible to intratumoral factors the limit efficacy of conventional Tcell therapies. In other embodiments CAR-DC are targeting to tumorantigens.

In one embodiment of the invention, protocols similar to Kershaw et al.are utilized with the exception that tumor endothelial antigens aretargeted as opposed to conventional tumor antigens. Such tumorendothelial antigens include CD93, TEM-1, VEGFR1, and survivin.Antibodies can be made for these proteins, methodologies for which aredescribed in U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, and5,639,641. In one example that may be utilized as a template forclinical development, T cells with reactivity against the ovariancancer-associated antigen alpha-folate receptor (FR) were generated bygenetic modification of autologous T cells with a chimeric geneincorporating an anti-FR single-chain antibody linked to the signalingdomain of the Fc receptor gamma chain. Patients were assigned to one oftwo cohorts in the study. Eight patients in cohort 1 received a doseescalation of T cells in combination with high-dose interleukin-2, andsix patients in cohort 2 received dual-specific T cells (reactive withboth FR and allogeneic cells) followed by immunization with allogeneicperipheral blood mononuclear cells. Five patients in cohort 1experienced some grade 3 to 4 treatment-related toxicity that wasprobably due to interleukin-2 administration, which could be managedusing standard measures. Patients in cohort 2 experienced relativelymild side effects with grade 1 to 2 symptoms. No reduction in tumorburden was seen in any patient, Tracking 111In-labeled adoptivelytransferred T cells in cohort 1 revealed a lack of specific localizationof T cells to tumor except in one patient where some signal was detectedin a peritoneal deposit. PCR analysis showed that gene-modified T cellswere present in the circulation in large numbers for the first 2 daysafter transfer, but these quickly declined to be barely detectable 1month later in most patients. Similar CAR-T clinical studies have beenreported for neuroblastoma, B cell malignancies, melanoma, ovariancancer, renal cancer, mesothelioma, and head and neck cancer.

In one embodiment of the invention, PBMCs are derived from leukapheresisand CD14 monocytes are collected by MACS. After 3 days of culture, M-CSFat 100 ng/ml plasmid encoding the chimeric CAR-DC recognizingtumor-endothelium specific antigen and subsequently selected for geneintegration by culture in G418. In another embodiment the generation ofdual-specific T cells is performed, stimulation of allogeneic monocyticcells is achieved by coculture of patient PBMCs with irradiated (5,000cGy) allogeneic donor PBMCs from cryopre-served apheresis product (mixedlymphocyte reaction). The MHC haplotype of allogeneic donors isdetermined before use, and donors that differed in at least four MHCclass I alleles from the patient are used. Culture medium consisted ofAimV medium (Invitrogen, Carlsbad, Calif.) supplemented with 5% humanAB⁻ serum (Valley Biomedical, Winchester, Va.), penicillin (50units/mL), streptomycin (50 mg/mL; Bio Whittaker, Walkersville, Md.),amphotericin B (Fungizone, 1.25 mg/mL, Biofluids, Rockville, Md.),L-glutamine (2 mmol/L; Mediatech, Herndon, Va.), and human recombinantIL-2 (Proleukin, 300 IU/mL; Chiron). Mixed lymphocyte reaction consistedof 2×10⁶ patient monocytes and 1×10⁷ allogeneic stimulator PBMCs in 2 mLAimV per well in 24-well plates. Between 24 and 48 wells are culturedper patient for 3 days, at which time transduction is done by aspirating1.5 mL of medium and replacing with 2.0 mL retroviral supernatantcontaining 300 IU/mL IL-2, 10 mmol/L HEPES, and 8 μg/mL polybrene(Sigma, St. Louis, Mo.) followed by covering with plastic wrap andcentrifugation at 1,000×g for 1 hour at room temperature. Afterovernight culture at 37° C./5% CO₂, transduction is repeated on thefollowing day, and then medium was replaced after another 24 hours.Cells are then resuspended at 1×10⁶/mL in fresh medium containing 0.5mg/mL G418 (lnvitrogen) in 175-cm² flasks for 5 days before resuspensionin media lacking G418. Cells are expanded to 2×10⁹ and then restimulatedwith allogeneic PBMCs from the same donor to enrich for T cells specificfor the donor allogeneic haplotype. Restimulation is done by incubatingpatient T cells (1×10⁶/mL) and stimulator PBMCs (2×10⁶/mL) in 3-literFenwall culture bags in AimV+additives and IL-2 (no G418). Cell numberswere adjusted to 1×10⁶/mL, and IL-2 was added every 2 days, untilsufficient numbers for treatment were achieved.

The present invention relates to a strategy of adoptive cell transfer ofmonocytes or DC transduced to express a chimeric antigen receptor (CAR).CARs are molecules that combine antibody-based specificity for a desiredantigen (e.g., tumor endothelial antigen) with a T cellreceptor-activating intracellular domain to generate a chimeric proteinthat exhibits a specific anti-tumor endothelium cellular immuneactivity. In one embodiment the present invention relates generally tothe use of monocytes or DC cells genetically modified to stably expressa desired CAR that possesses high affinity towards tumor associatedendothelium. Monocytes or DC cells expressing a CAR are referred toherein as CAR-DC cells or CAR modified DC cells. Preferably, the cellcan be genetically modified to stably express an antibody binding domainon its surface, conferring novel antigen specificity that is MHCindependent. In some instances, the monocyte or DC cell is geneticallymodified to stably express a CAR that combines an antigen recognitiondomain of a specific antibody with an intracellular domain of theCD3-zeta chain or Fc.gamma.RI protein into a single chimeric protein. Inanother embodiment, TLR signaling molecules are engineered in theintracellular portion of the CAR, said molecules include TRIF, TRADD,and MyD99. In one embodiment, the CAR of the invention comprises anextracellular domain having an antigen recognition domain, atransmembrane domain, and a cytoplasmic domain. In one embodiment, thetransmembrane domain that naturally is associated with one of thedomains in the CAR is used. In another embodiment, the transmembranedomain can be selected or modified by amino acid substitution to avoidbinding of such domains to the transmembrane domains of the same ordifferent surface membrane proteins to minimize interactions with othermembers of the receptor complex. Preferably, the transmembrane domain isthe CD8a hinge domain.

With respect to the cytoplasmic domain, the CAR of the invention can bedesigned to comprise the CD80 and/or CD86 and/or CD40L and/or OX40Lsignaling domain by itself or be combined with any other desiredcytoplasmic domain(s) useful in the context of the CAR of the invention.In one embodiment, the cytoplasmic domain of the CAR can be designed tofurther comprise the signaling domain of MyD88. For example, thecytoplasmic domain of the CAR can include but is not limited to CD80and/or CD86 and/or CD40L and/or OX40L signaling modules and combinationsthereof. In another embodiment of the invention inhibition of TGF-betais performed either by transfection with an shRNA possessing selectivelytowards TGF-beta or by constructing the CAR to possess a dominantnegative mutant of TGF-beta receptor. This would render the CAR-DC cellresistant to inhibitory activities of the tumors. Accordingly, theinvention provides CAR-DC cells and methods of their use for adoptivetherapy. In one embodiment, the CAR-DC cells of the invention can begenerated by introducing a lentiviral vector comprising a desired CAR,for example a CAR comprising anti-CD19, CD8α hinge and transmembranedomain, and MyD88, into the cells. The CAR-DC cells of the invention areable to replicate in vivo resulting in long-term persistence that canlead to sustained tumor control.

One skilled in the art will appreciate that these methods, compositions,and cells are and may be adapted to carry out the objects and obtain theends and advantages mentioned, as well as those inherent therein. Themethods, procedures, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. It will be apparent to one skilled in the artthat varying substitutions and modifications may be made to theinvention disclosed herein without departing from the scope and spiritof the invention. Those skilled in the art recognize that the aspectsand embodiments of the invention set forth herein may be practicedseparate from each other or in conjunction with each other. Therefore,combinations of separate embodiments are within the scope of theinvention as disclosed herein. All patents and publications mentioned inthe specification are indicative of the levels of those skilled in theart to which the invention pertains. All patents and publications areherein incorporated by reference to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions indicates the exclusion ofequivalents of the features shown and described or portions thereof. Itis recognized that various modifications are possible within the scopeof the invention disclosed. Thus, it should be understood that althoughthe present invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the disclosure.

1.-20. (canceled)
 21. A modified macrophage or a monocyte comprising achimeric antigen receptor (CAR), wherein the CAR comprises an anti-HER2antigen binding domain, a transmembrane domain, and an intracellulardomain of a stimulatory and/or co-stimulatory molecule.
 22. The modifiedmacrophage or a monocyte of claim 21, wherein the CAR further comprisesa hinge domain.
 23. The modified macrophage or a monocyte of claim 22,wherein the hinge domain comprises a CD8 hinge domain.
 24. The modifiedmacrophage or a monocyte of claim 21, wherein the transmembrane domaincomprises a CD8 transmembrane domain.
 25. The modified macrophage or amonocyte of claim 21, wherein the intracellular domain comprises CD80and/or CD86 and/or CD40L and/or OX40L signaling domains.
 26. Themodified macrophage or a monocyte of claim 21, wherein the intracellulardomain comprises a CD3 zeta intracellular domain or an intracellulardomain capable of activating TLR4 signaling.
 27. The modified macrophageor a monocyte of claim 21, wherein the antigen binding domain isselected from a group consisting of a chimeric antibody, syntheticantibody, humanized antibody, single heavy chain antibody, single lightchain antibody, scFv, and antigen-binding fragments thereof.
 28. Themodified macrophage or a monocyte of claim 21, wherein the modifiedmacrophage or a monocyte expresses HLA DR.
 29. The modified cell ofclaim 21, wherein the modified macrophage or a monocyte is a humanmacrophage or a monocyte.
 30. The modified macrophage or a monocyte ofclaim 21, further comprising linkers linking the transmembrane domain tothe antigen binding domain.
 31. The modified macrophage or a monocyte ofclaim 21, further comprising an agent, wherein the agent is selectedfrom the group consisting of a nucleic acid, a peptide and anycombination thereof.
 32. A pharmaceutical composition comprising themodified macrophage or a monocyte of claim 21 and a pharmaceuticallyacceptable carrier.
 33. A method of treating a malignancy in a subject,comprising: administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising a modified macrophageor a monocyte comprising a chimeric antigen receptor (CAR), wherein theCAR comprises an antigen binding domain, a transmembrane domain and anintracellular domain of a stimulatory and/or co-stimulatory molecule,wherein the antigen binding domain targets a tumor antigen.
 34. Themethod of claim 33, wherein the tumor antigen is Receptortyrosine-protein kinase ERBB2 (HER2).
 35. The method of claim 33,wherein the malignancy is a carcinoma, a sarcoma, a leukemia, ormelanoma.
 36. The method of claim 33, wherein the antigen binding domainof the CAR comprises an antibody selected from the group consisting of achimeric antibody, synthetic antibody, humanized antibody, single heavychain antibody, single light chain antibody, scFv, and antigen-bindingfragments thereof.
 37. The method of claim 33, wherein the transmembranedomain of the CAR comprises a CD8 transmembrane domain.
 38. The methodof claim 33, wherein the intracellular domain of the CAR comprises CD80and/or CD86 and/or CD40L and/or OX40L signaling domains.
 39. The methodof claim 33, wherein the intracellular domain of the CAR comprises a CD3zeta intracellular domain.
 40. The method of claim 33, wherein themodified macrophage or a monocyte exhibits targeted effector activity.41. The method of claim 40, wherein the targeted effector activity isdirected against a target cell comprising the tumor antigen.
 42. Themethod of claim 40, wherein the targeted effector activity is selectedfrom the group consisting of phagocytosis, cytotoxicity, and antigenpresentation.
 43. The method of claim 40, wherein the targeted effectoractivity is enhanced by inhibition of CD47 or signal-regulatory proteinα activity.
 44. The method of claim 33, wherein the pharmaceuticalcomposition further comprises an agent, wherein the agent is selectedfrom the group consisting of a nucleic acid, a peptide and anycombination thereof.
 45. The method of claim 33, wherein >75% of themodified cells in the pharmaceutical composition express the CAR orwherein the modified human monocytes in the population of cells aregenetically modified to stably express the CAR.
 46. The method of claim33, wherein the modified macrophage or a monocyte is obtained from theindividual patient to be treated or allogeneic to the patient.
 47. Themethod of claim 33, wherein the modified macrophages or monocytes areadministered by intratumoral injection.
 48. The method of claim 33,wherein the modified macrophages or monocytes are administeredintratumorally.
 49. The method of claim 33, wherein the modifiedmacrophage or monocyte is administered to a patient in conjunction withat least one other treatment modality.
 50. The method of claim 49,wherein the at least one other treatment modality comprises an agentthat induces immunogenic cell death; chemotherapy; and/or immunemodulators including BCG, CpG DNA, interferon alpha, tumor bacterialtherapy, checkpoint inhibitors, Treg depleting agents, and low dosecyclophosphamide.